Each protein in a living cell will be cleaved by a protease (Doucet et al., 2008; Lopez-Otin and Bond, 2008). The purpose of these enzymatic events ranges from shredding damaged proteins that might otherwise harm the cell, to sculpting signal precursors to initiate cell communication (Lopez-Otin and Bond, 2008). Aside from controlling essential processes in all forms of life, protease inhibition has proven to be a particularly effective therapeutic strategy, especially in hypertension and antiviral treatment (Drag and Salvesen, 2010).
Ultimately deciphering how a protease shapes the signaling characteristics of healthy cells, or targeting it for therapeutic intervention in disease, requires a sophisticated understanding of its enzymatic properties. Kinetic dissection of protease catalysis has been key in revealing these properties (Huntington, 2012; Perona and Craik, 1997; Timmer et al., 2009). Coupled with structural analyses, these studies have established that both cytosolic and extracellular proteases are designed to bind their substrates specifically at discrete sites, with affinity reflected in the Michaelis constant (KM), and endowed with catalytic residues that function in rate enhancement, reflected in the turnover number (kcat). The catalytic efficiency of a protease is the quotient of these two parameters, and typically ranges from 104-107M−1s−1 (108 reflects enzymes whose activity is limited by diffusion).
Intramembrane proteases, in contrast to these well-studied soluble proteases, are a more recently-discovered class of extraordinary enzymes that evolved independently to catalyze hydrolysis immersed within the membrane (De Strooper and Annaert, 2010; Fluhrer et al., 2009; Makinoshima and Glickman, 2006; Urban and Dickey, 2011; Wolfe, 2009). Despite this complexity, there is significant incentive for understanding how proteolysis is accomplished within these constraints, because intramembrane proteases hold great promise for developing therapies: rhomboid proteases are implicated in Parkinson's disease and parasite invasion (Urban and Dickey, 2011); γ-secretase in Alzheimer's disease and leukemia (De Strooper and Annaert, 2010; Wolfe, 2009); signal peptide peptidases in immunity and hepatitis C virus assembly (Fluhrer et al., 2009); and site-2 proteases in the virulence of some of the world's deadliest bacterial and fungal pathogens (Makinoshima and Glickman, 2006; Urban, 2009).
Major insights into the molecular architecture of these remarkable enzymes has been gained from a series of high-resolution intramembrane protease crystal structures of prokaryotic orthologs (Li et al., 2013; Wolfe, 2009), as led by analyses of the Escherichia coli rhomboid protease GlpG (as summarized in (Urban, 2010). In contrast, analysis of catalysis within the membrane in quantitative terms has not been achieved with any intramembrane protease, making it difficult to decipher their functional properties. Current models are based largely on extrapolations from soluble proteases, which evolved independently and could be different. In fact, the membrane is a fundamentally unusual setting for proteolysis: chemically, the membrane is viscous and excludes water, which is both essential for proteolysis and affects how proteins interact. Spatially, proteins in a membrane exist in a two-dimensional plane and are orientationally-confined relative to each other. Although techniques for studying proteins inside the membrane are scarce, understanding the consequences of this environment, and how intramembrane proteases function within it, requires interrogating the kinetics of proteolysis within its natural membrane setting.
We have overcome multiple inherent limitations to develop the first ‘inducible’ membrane reconstitution system for the quantitative analysis of rhomboid proteolysis occurring within the membrane and in real time. The results reveal that, contrary to expectations, rhomboid proteolysis is a slow reaction that is not driven by affinity of enzyme for substrate. Instead, these insights suggest a completely different mode of action for this ancient and widespread family of enzymes.